Transcriptional alterations in bladder epithelial cells in response to infection with different morphological states of uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) may undergo a cyclic cascade of morphological alterations that are believed to enhance the potential of UPEC to evade host responses and re-infect host cell. However, knowledge on the pathogenic potential and host activation properties of UPEC during the morphological switch is limited. Microarray analysis was performed on mRNA isolated from human bladder epithelial cells (HBEP) after exposure to three different morphological states of UPEC (normal coliform, filamentous form and reverted form). Cells stimulated with filamentous bacteria showed the lowest number of significant gene alterations, although the number of enriched gene ontology classes was high suggesting diverse effects on many different classes of host genes. The normal coliform was in general superior in stimulating transcriptional activity in HBEP cells compared to the filamentous and reverted form. Top-scored gene entities activated by all three morphological states included IL17C, TNFAIP6, TNF, IL20, CXCL2, CXCL3, IL6 and CXCL8. The number of significantly changed canonical pathways was lower in HBEP cells stimulated with the reverted form (32 pathways), than in cells stimulated with the coliform (83 pathways) or filamentous bacteria (138 pathways). A host cell invasion assay showed that filamentous bacteria were unable to invade bladder cells, and that the number of intracellular bacteria was markedly lower in cells infected with the reverted form compared to the coliform. In conclusion, the morphological state of UPEC has major impact on the host bladder response both when evaluating the number and the identity of altered host genes and pathways.

Urinary tract infection (UTI) is the second most common infectious disease in humans, following infections in the respiratory tract, and affects mainly women 1 . One reason for the high frequency of UTIs is that a large proportion (approximately 25-35%) of women treated for UTI will subsequently be afflicted by a recurrent infection within 6 months after the initial infection 1,2 . Uropathogenic Escherichia coli (UPEC) are responsible for the vast majority of UTIs in uncompromised patients and recurrent UTI is often caused by the same clone as the first infection 3,4 . This suggests that UPEC have evolved strategies to persist in the urinary tract and evade antibiotic treatment and host response mechanisms.
It is now well established that UPEC strains are able to invade host bladder epithelial cells where they can replicate and form biofilm-like intracellular bacterial communities. Studies in mouse bladders have suggested that UPEC undergo a cyclic cascade in which intracellular UPEC replicate and subsequently escape from the colonized bladder cells to enter a new cycle of infection [5][6][7] . The subsequent resurgence of these intracellular reservoirs is considered a possible cause of recurrent or chronic UTI 8 . Intracellular UPEC undergo morphological alterations characterized by transformation from a rod shape to a coccoid morphology that may follow by an additional transition into a filamentous form 6,7,9 . The filamentous, elongated morphology dominates during escape from the host bladder cells 7 , but re-infection of host cells is believed to occur first after reverting of Advanced Cell Systems AG, Bern, Switzerland) from a single donor was cultured in CnT-58 cell culture medium (CELLnTEC) supplemented with 100 U/mL penicillin and 100 μL/mg streptomycin (PEST) (Invitrogen Ltd, Paisley, UK) in a humidified atmosphere with 5% CO 2 at 37 °C. At confluency the culture was differentiated into umbrella cell-like cells during four days using CnT-21 differentiation medium (CELLnTEC) supplemented with 1 mM CaCl2 in 6-well plates. The ESBL-producing E. coli (designated ESBL019) was obtained from the bacterial culture collection at Örebro University hospital, Sweden. Antimicrobial susceptibility testing was performed as recommended by the Swedish reference Group for Antibiotics (www. srga. org). This isolate has previously been characterized and determined to belong to the ST131 clone and to be multidrug resistant (CTX-M-15), including resistance to ceftibuten 19 . Bacteria were maintained on tryptic soy agar (Becton, Dickinson and Company, Sparks, MD). Prior to experiments, bacteria were inoculated in Difco Luria-Bertani (LB) broth (Lennox; Franklin Lakes, NJ, USA) and incubated at 37 °C aerobically on a shaker overnight. Bacteria were suspended in sterile phosphate buffered saline (PBS) prior to inoculation of cell culture medium (CCM) with or without ceftibuten (480 ng/mL).
Cell stimulation. Three different morphologies of ESBL019 were prepared for infecting HBEP cells as previously described 15 . Briefly, ESBL019 Coliform was grown for 3 h in cell culture medium (CCM) without ceftibuten, ESBL019 Filamentous was grown for 3 h in the presence of ceftibuten (480 ng/ml) and ESBL019 Reverted was grown in the presence of ceftibuten (480 ng/ml) for 3 h and then without ceftibuten for 1 h to allow its reversion. The bacteria were thereafter added to HBEP cells, in 6-well plates, in the presence (ESBL019 Filamentous) or absence (ESBL019 Coliform, ESBL019 Reverted) of ceftibuten (480 ng/ml). The protocol and the reversible cell morphology transition are shown in Fig. 1.
Prior to inoculation, the bacterial concentrations of all the morphologies were adjusted in order to infect HBEP cells with MOI 10. Infected HBEP cells were incubated for 4 h in 6-well plates in a humified atmosphere with 5% CO 2 at 37 °C after which supernatants and RNA were collected. Alterations in HBEP gene expression in response to the different morphological states of ESBL019 were evaluated in comparison to unstimulated control cells incubated in CCM without ceftibuten. The bacteria from these experiments were harvested and used for transcriptional analysis as reported in a previous study 15 . RNA preparation and microarray. Total RNA from infected and unstimulated HBEP cells was isolated with RNeasy Mini Kit (Qiagen Technologies, Hilden, Germany) according to manufacturer instructions. RNA concentration and purity were measured using a Nano-Drop ND-1000 Spectrophotometer (Nano-Drop Technology Inc., Wilmington, DE, USA). All samples had OD260/280 and OD260/230 ratios above 1.9. RNA quality was further assessed using the Agilent RNA 6000 Nano Kit (Agilent) with 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer's guidelines. RNA integrity number (RIN) values were > 9.0 for all samples. High-quality total RNA was used to prepare amplified Cy3-labeled cRNA with the Data processing and statistical analysis. Microarray data analysis was performed using GeneSpring GX version 11.5 (Agilent) after per chip and 75th percentile shift gene normalization of samples (n = 4). Differentially expressed genes were subjected to Gene Ontology enrichment analysis in GeneSpring and to functional analyses through Ingenuity Pathway Analysis (IPA, QIAGEN Inc., https:// www. qiage nbioi nform atics. com/ produ cts/ ingen uityp athway-analy sis) with Core analyses for enrichments in canonical pathway, downstream functions and upstream regulators. For analyzing differences between groups one-way analysis of variance (ANOVA) parametric test was used. Significant entities (q < 0.05) were obtained by Tukey HSD post hoc test followed by Bonferroni multiple testing correction and a fold change cut-off ≥ 2. Significant GO term enrichment was set at a p-value < 0.05.

Results
Gene expression alterations in response to different morphological states of ESBL019. Microarray analysis was performed on mRNA isolated from HBEP cells exposed to bacteria following their morphological transition from coli form to a filamentous form and when reverted to their original coli form following antibiotic withdrawal. In total 2018 entities were differentially expressed (p < 0.05) with at least a ≥ twofold change compared to unstimulated control cells. All three morphological states of the bacteria, Coliform, Filamentous and Reverted, induced definite changes in gene expressions in HBEP cells with 737, 374 and 634 up www.nature.com/scientificreports/ regulated and 597, 55 and 620 down-regulated gene expressions, respectively (Table 1). Differentially expressed gene entities within each group are visualized using a Venn diagram (Fig. 2). When comparing unstimulated HBEP cells incubated with or without ceftibuten only a few, in comparison to the number of differences seen with bacterial exposure, were seen. In total 72 entities were differentially expressed and of these 50 were entities annotated to uncharacterized locus areas and hypothetical proteins. The remaining 22 entities were in general transcript variants annotated to genes with a low fold change and not included among genes found in relation to bacterial infection. Taken together 341 altered gene entities are shared between Coliform and Filamentous (R1 + R2) with 305 upregulated and 36 down-regulated entities, and 165 gene entities were shared between Filamentous and Reverted (R2 + R3) with 138 up-regulated and 27 down-regulated entities. In addition, 652 gene entities were shared between Coliform and Filamentous (R2 + R4) with 342 up-regulated and 310 down-regulated entities ( Table 1, Fig. 2). A total of 159 gene entities were shared between all three groups with 135 up-regulated and 24 downregulated entities (Fig. 2). Here, we identified a set of strongly up-regulated genes with a fold change > 100, including IL17C, TNFAIP6, TNF, IL20, CXCL2, CXCL3, IL6, CXCL8, ICAM1, CXCL1, IL23A, CSF3, CCL20, CSF2, PTX3 (Table 2). Up-regulation of these genes, except for ICAM1, was markedly higher in HBEP cells stimulated with the Coliform compared to cells stimulated with Filamentous and Reverted bacteria ( Table 2). For a summary of the altered unique and shared gene entities see Supplement (Tables S1-S6).
Gene ontology analysis. Gene ontology analysis was performed on differentially expressed entities after stimulation with bacteria from each of the three morphological states. In total, 256 (Coliform), 387 (Filamentous) and 286 (Reverted) significantly (p < 0.05) enriched gene ontologies were identified. Of these, 18 (Coliform), 67 (Filamentous) and 27 (Reverted) gene ontologies were uniquely enriched (Tables S7-S9). Two gene ontologies (positive regulation of leukocyte chemotaxis and negative regulation of cell proliferation) were enriched in all three morphological states (Table S13). The number of enriched gene ontologies shared between two morphological states was 6 (Coliform + Filamentous), 9 (Coliform + Reverted) and 5 (Filamentous + Reverted) (Tables S10-S12).  Fig. 3A. TNF, IL1B and IL1A are differently activated genes that are also identified here as key upstream regulators of gene expression. The predicted activation score of the top-scored upstream regulators was in general lower for cells stimulated with Reverted bacteria than for the other two morphologies (Fig. 3A). Analysis of upstream regulators for gene entities that were uniquely altered i.e., in response to only one of the morphological states are shown in Fig. 3B. These data demonstrate that the activation score pattern for some regulators is comparable but that many of the regulators show a specific activation score pattern for the group of uniquely altered gene entities associated with the respective morphological states. The upstream regulators that are predicted to regulate gene expression of NLRP3 (only significantly increased in HBEP cells stimulated with the Coliform) and CXCL8 (significantly increased in all three morphological states) are shown in Fig. 4. A general observation for these genes is that the number of significantly altered upstream regulators, as well as their predicted activation of the respective genes, are lower in HBEP stimulated with the Filamentous form.
Pathway analysis. A canonical pathway analysis was performed for enrichment of altered gene entities in functional pathways with a cut-off of at least two matched entities in each pathway. Curation for exclusion of redundant pathways was made. The number of significantly changed canonical pathways was higher in HBEP cells stimulated with Filamentous bacteria (138 pathways), than in cells stimulated with the Coliform (83 pathways) or Reverted form (32 pathways). The top-scored significantly changed canonical pathways in HBEP cells stimulated with the different morphologies are shown in Fig. 5. Most of the pathways was activated (positive z-score), although PPAR and erythropoietin signalling pathway showed decreased activity in cells stimulated with all three morphologies (Fig. 5). Noteworthy, pathways related to IL-17/IL-17A/IL-17F-signalling, TREM1 and HMBG1 signalling were found among the top-scored pathways in all of the morphological states.

Invasion of host bladder cells.
The ability of the different bacterial morphologies to invade bladder epithelial cells was assessed by quantifying the number of intracellular bacteria after lysis of cells. Intracellular bacteria were detected in all experiments using Coliformed bacteria (13 ± 3.7 CFU/well). However, filamentous bacteria were unable to invade bladder cells, and the number of intracellular bacteria was markedly lower in cells infected with the Reverted form compared to the Coliform (Fig. 6).

Discussion
UPEC pathogenesis involves a cyclic transition between extra-and intracellular localisation, along with a synchronized transition to filaments that subsequently reverts back to normal rod-shaped bacteria with a potential to re-infect host bladder cells 5,7 . In addition, and as applied in the present study, an extensive formation of www.nature.com/scientificreports/ bacterial filaments can also occur as a result of sub-lethal concentrations of antibiotics [13][14][15] . Previous studies have mainly focused on characteristics of the different bacterial morphologies 15,18 , while the host cell response following infection with the different morphologies have not been determined. In the present study we used human primary bladder epithelial cells (HBEP), instead of more commonly used bladder cancer epithelial cell lines, in order to increase the biological relevance of the host-bacteria interactions. However, a limitation using this one donor cell line is that the data is based on the cell responses from one donor, which may be different from the cell response obtained from different donors.  www.nature.com/scientificreports/ stimulated with Coliform and Reverted bacteria. This suggests that Filamentous bacteria have diverse effects and affect a variety of different classes of host genes. Overall, the majority of the identified changes in gene ontologies were associated with immune system processes, cell signalling and cytokine/chemokine production, which is an expected finding. Two gene ontologies, positive regulation of leukocyte chemotaxis and negative regulation of cell proliferation, were enriched in cells stimulated with all three morphological states. These gene ontologies include many genes that are associated with host immune responses 20,21 such as CXCL1, CXCL2, CXCL3, CXCL8, IL-6, IL17C and TNF, demonstrating that all morphological states can evoke a host immune response of bladder epithelial cells. However, it was evident from analysis of the transcriptional activity of the individual genes that the degree of host activation markedly differed between the three morphological states. The normal Coliform was often superior in stimulating transcriptional activity in HBEP cells compared to the Filamentous and Reverted form. UPEC depend on fimbriae for adhesion and invasion of bladder epithelial cells 22 , but the decreased activation of host immune factors found in the present study is unlikely to be explained by decreased adhesion of Filamentous and Reverted bacteria to HBEP cells. Filamentous and/or Reverted UPEC appear to maintain their functional adhesion properties 9,10,15 , and up-regulate P-and type-1 fimbriae-associated genes 15 , suggesting that the morphological switch do not attenuate the ability of the bacteria to adhere to host cells. Nevertheless, although the Filamentous and Reverted UPEC can adhere there are other factors of importance for host activation that may be depressed. We found in the present study that the Filamentous form was unable to invade host bladder cells. This emphasizes that the filamentous phase of UPEC infection needs to be transient and reversible given that a secondary host cell re-infection is believed to occur first after reverting of filaments to a rod-shaped morphology 10 . In our experimental set-up, the Reverted form was evaluated 1 h after antibiotic removal when the bacteria, based on microscopical inspection, appeared to have retained their normal rod-shaped form. However, it is obvious that the Reverted form in our study is rather in a transitory state than fully reverted given its compromised host-activating properties, including a low host invasion capacity. Thus, the overall lower transcriptional activation evoked by Filamentous and Reverted UPEC may be related to the impaired host cell invasion capacity of these morphological forms. The endotoxin LPS mediates multiple aspects of UPEC pathogenesis by interacting with the Toll-like receptor 4 (TLR4), which subsequently activates the innate immune response and release proinflammatory cytokines 23 .    www.nature.com/scientificreports/ Our previous study demonstrated that genes associated with LPS biosynthesis were up-regulated in the Filamentous bacteria, but down-regulated in Reverted bacteria 15 . In the present study, analysis of upstream regulators revealed a lower predicted activation z-score for TLR4 in HBEP cells stimulated with Reverted bacteria, while the activation score was comparable in cells stimulated with Coliform and Filamentous bacteria. HBEP is a low LPS-responsive cell line (unpublished observations) and, in combination with a variable expression of LPS and TLR4 among the morphological states, it is not likely that the depressed host activation by Filamentous and Reverted bacteria are explained by alterations in TLR4 signalling. Interestingly, the adhesion molecule ICAM-1 showed a higher activity in cells stimulated by Filamentous than Coliform bacteria. UPEC enhance the expression of ICAM-1 on urinary tract epithelial cells to facilitate neutrophil migration 24 , and a more pronounced physical interaction between the elongated filaments and host cells 9,10,15 may possibly trigger an increased transcription of ICAM-1 by HBEP cells.
The cell division gene damX 18 and SulA, an effector molecule in the bacterial SOS response 9 , have been implicated as a key inducer of UPEC filamentation. Deletion of damX or sulA abrogated both UPEC filamentation and the ability to establish murine urinary tract infection 9,18 , suggesting that the ability to transiently switch into alternative morphologies may be an advantage for uropathogenesis. Our previous study has shown that ceftibuten-induced UPEC filamentation is associated with increased expression of sulA 15 , demonstrating that the filaments formed by ceftibuten in vitro show similarities with the morphological alterations that occur in vivo. A weak activation of the host immune system, as noted for Filamentous and Reverted bacteria, may be considered as an advantage for the bacteria since migration of phagocytic cells, like neutrophils, depend on an activated host response and secreted chemokines 20,21 . UPEC have evolved different mechanisms to suppress host recognition and activation e.g., by secreting TcpC that binds to MyD88 and inhibits down-stream signalling and cytokine responses by host cells in the urinary tract 25 . However, the expression of tcpC was found to be significantly downregulated in the Filamentous and Reverted forms compared to the normal Coliform 15 , which argues against an involvement of TcpC. Nevertheless, it cannot be excluded that the weak stimulatory effect on cytokine gene expression demonstrated in the present study by the Filamentous and Reverted forms involves an active suppression of host activation. Pro-inflammatory soluble cytokines that are generated by immune system cells in response to bacterial infection may also, through autocrine and paracrine mechanisms, act as upstream regulators of gene expression 26 . Our data showed that the cytokines TNF, IL1B and IL1A, which were all differently activated gene entities, were highly ranked among the upstream regulators. Thus, the overall lower primary activation of many HBEP genes in response to Filamentous and Reverted bacteria may, therefore, also affect the capacity of these cytokines to act as upstream regulators of other target molecules. Taken together, the results suggest that the Filamentous and Reverted morphological states evoke an overall weaker host immune response than the normal Coliform, but the underlying mechanisms needs to be further elucidated.
Some of the top-scored canonical pathways such as IL-6, TLR and IL-8 signalling are all pathways with an established connection to mechanisms underlying UTI pathogenesis 20,21 . Interestingly, several pathways related to IL-17-signalling were among the top-scored pathways in all three morphological states. The importance of IL-17A for innate immunity in UTI is demonstrated by a defect in acute clearance of UPEC and higher bacterial burden in IL-17A-deficient mice 27,28 . Also, the known action of IL-17 in cell recruitment and immune regulation 29 imply that this cytokine is required to fine tune innate cellular defenses to UPEC in the bladder. IL-17C was the most upregulated gene in cells stimulated with Coliform and Filamentous bacteria, while a considerably lower expression was noted in response to Reverted bacteria. This is supported by data from the canonical pathway analysis demonstrating a considerable higher activation of ¨IL-17 Signaling¨ in Coliform and Filamentous bacteria than in the Reverted form. The danger-associated molecules high-mobility group box 1 (HMGB1) signalling and triggering receptor expressed on myeloid cells 1 (TREM1) signalling were other canonical pathways that displayed high activation scores. HMGB1 has many functions in the cell, including a proinflammatory role when released into the extracellular environment by passive release from necrotic cells or in an active manner following cell stimulation with e.g., LPS, IL-1β and TNF-α 30 . In the urinary tract, HMGB1 has been mainly been associated with the progression of urothelial carcinoma 31 , but bacterial infection of the urinary tract also stimulates increased expression of HMGB1 32 . Besides its own direct intracellular signaling pathway, TREM1 activation cross-talks with intracellular signalling pathways of several TLRs and can thereby contribute to and amplify the magnitude of inflammation 33 . A soluble form of TREM1 is shed from host cells and several studies have investigated the potential use of TREM-1 as a diagnostic marker for UTI 34,35 . Taken together, the pathway analysis demonstrated that the number of enriched pathways, and often the activation scores, were lowest in HBEP cells stimulated by the Reverted form. The Reverted form has undergone intense morphological transitions which appear to result in diminishing host activating properties. Both the number of significantly changed canonical pathways and gene ontology classes was highest in HBEP cells stimulated with Filamentous bacteria. Hence, the filamentous form may due to its exaggerated size be attributed to enhanced host recognition and surveillance. Moreover, our previous findings have shown that the filamentation process per se is associated with increased release of endotoxins and ATP into the cell supernatant, and that these soluble bacterial products are able to stimulate cytokine secretion from bladder epithelial cells 14 . Thus, the widespread activating response caused by the filamentous form may involve enhanced release of various extracellular host activating factors during the morphological transition into the filamentous form.
In conclusion, we have shown that the host bladder response to infection with different morphological states of UPEC, proposed to replicate the uropathogenic cascade, exhibit differences both in the number and identity of altered host genes and pathways. Re-infection of host bladder cells, as a potential cause of recurrent UTI, is likely to be a complex process and the outcome controlled both by the transient morphological state of the bacteria and the host immune response.

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary  Information files).